DESCRIPTION: This is an amended proposal to continue a study of the mechanisms that are responsible for the stable, long-term maintenance of homeotic gene expression during Drosophila development. It focuses on the trithorax gene, which has been shown to be required for the maintenance of the expression patterns of the genes of the BX-C and ANTP-C and to counterbalance the effects of the Polycomb group genes, which act as negative regulators of homeotic gene expression. Through a detailed genetic and molecular genetic analysis of the trx gene, and through a detailed functional dissection of the trx protein, Dr. Harte proposes an ambitious experimental attack on questions whose resolution likely involves the relationship between chromatin conformation and state of transcription activation. This is an aspect of transcriptional regulation that has fascinated biologists for many years, and remains poorly understood, but is likely to attract increasing interest during the next several years. During the previous grant period, a significant effort was devoted to a detailed description of the varied responses to trx mutation by numerous genes and to the cloning and molecular characterization of the trx gene. The trx transcription unit was found to be large and to produce a complex array of RNA products and proteins that are estimated to range in size between 360 and 420 kd. The heroic efforts to characterize this gene (including transformation with rescue constructs containing a 34 kb genomic fragment) have been rewarded by the discovery of several recognizable sequence domains in the protein coding sequence, including a presumed steroid receptor family DNA binding domain and a region homologous to the human ALL-1 proteins which have been implicated in childhood Acute Lymphocytic Leukemias. The trx protein has been shown to bind to DNA in vitro, to bind to 63 specific sites on polytene chromosomes, and to functionally associate with a related trx-G protein, ash-1, and functional trx response elements have been identified. The experimental goals of the proposed research constitute an extensive and detailed analysis of trx. High resolution mapping of trx chromosome binding sites will be pursued using germ line transformation of reporter constructs to map both chromosomal binding sites and the ability of these sequences to affect expression of linked genes. Characterization of in vitro DNA binding will be pursued, buoyed by the recent success in showing that a fragment of trx protein is active in a mobility shift assay with a 660 bp core PRE. These studies will be extended to better define binding affinities and specificities, and in collaboration with Renato Paro, to map sites to which trx protein binds in vivo. To identify other proteins with which trx associates, several approaches are described, including co-immunoprecipitation, further characterization of the apparent association between trx and ash-1, and use of the polytene binding assay in various mutant backgrounds. Trx will be overexpressed with heat shock transgenic constructs and by increasing its gene dosage to determine the effects on homeotic gene expression and to test models of competition between Pc-G and trx-G proteins. Finally, structure/function studies of the trx protein will be pursued by defining the mutations present among the large collection of extant trx alleles and by generating a variety of mutations in genes that will be transformed into the genome.